General method for plasmid construction using homologous recombination

We describe a general method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with > 80% efficiency. Quantitative data on the influence of DNA concentration and overlap length on the efficiency of recombination are presented. Using a simple procedure, plasmids were shuttled from yeast into E. coli for subsequent screening and large-scale plasmid preps. This simple method for plasmid construction has several advantages. (i) It bypasses the need for extensive PCR amplification and for purification, modification and/or ligation techniques routinely used for plasmid constructions. (ii) The method does not rely on available restriction sites, thus fragment and vector DNA can be joined within any DNA sequence. This enables the use of multifunctional cloning vectors for protein expression in mammalian cells, other yeast species, E. coli and other expression systems as discussed. (iii) Finally, the technology exploits yeast strains, plasmids and microbial techniques that are inexpensive and readily available.     

Finite element modeling of three-dimensional pulsatile flow in the abdominal aorta: relevance to atherosclerosis

The infrarenal abdominal aorta is particularly prone to atherosclerotic plaque formation while the thoracic aorta is relatively resistant. Localized differences in hemodynamic conditions, including differences in velocity profiles, wall shear stress, and recirculation zones have been implicated in the differential localization of disease in the infrarenal aorta. A comprehensive computational framework was developed, utilizing a stabilized, time accurate, finite element method, to solve the equations governing blood flow in a model of a normal human abdominal aorta under simulated rest, pulsatile, flow conditions. Flow patterns and wall shear stress were computed. A recirculation zone was observed to form along the posterior wall of the infrarenal aorta. Low time-averaged wall shear stress and high shear stress temporal oscillations, as measured by an oscillatory shear index, were present in this location, along the posterior wall opposite the superior mesenteric artery and along the anterior wall between the superior and inferior mesenteric arteries. These regions were noted to coincide with a high probability-of-occurrence of sudanophilic lesions as reported by Cornhill et al. (Monogr. Atheroscler. 15:13-19, 1990). This numerical investigation provides detailed quantitative data on hemodynamic conditions in the abdominal aorta heretofore lacking in the study of the localization of atherosclerotic disease.     

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.     

Effect of the administration of fish oil by gavage on activities of antioxidant enzymes of rat lymphoid organs

Expression of retinoblastoma (Rb) protein was immunohistochemically examined in laryngeal squamous cell neoplasias from 72 patients. Staining patterns were considered with reference to such prognostic factors as patient's age, histologic grade, tumour size and lymph node status, and 5-year survival rate. Rb protein negativity, either partial or complete, was noted in 28.8% of cases and was associated with a significantly lower 5-year survival rate, as well as with a higher likelihood of lymph node metastasis. This suggests that Rb alteration may be a prognostic indicator in patients with laryngeal carcinoma.     

Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival of S. aureus. An STM library was ultimately created in S. aureus RN6390, with Tn917 being used to create the transposon mutations. Pools of S. aureus RN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain of S. aureus. In wound and murine abscess infection models, the putP mutant was approximately 10-fold more attenuated than was wild-type strain RN6390. Another S. aureus strain transduced with the putP mutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into the putP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureus high-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.     

Sickle cell adhesion to laminin: potential role for the alpha5 chain

Sickle red blood cell (RBC) adhesion to the endothelium and to exposed, underlying subendothelial proteins is believed to contribute to vascular occlusion in sickle cell disease. Laminin, a major component of the subendothelium, supports significant adhesion of sickle, but not normal RBCs. The purpose of this study was to define the adhesive region for sickle RBCs within a human laminin preparation using a flow adhesion assay designed to mimic physiologic flow through postcapillary venules. Because sickle RBCs did not adhere to the common laminin contaminants entactin or collagen type IV, neither of these proteins are likely to contribute to the observed adhesion to laminin. Known adhesive regions of laminin neither supported nor inhibited sickle RBC adhesion to laminin, suggesting a mechanism of adhesion previously uncharacterized in other laminin adhesion studies. Moreover, sickle RBCs did not adhere to mouse EHS laminin or to human laminin-2 (merosin), eliminating the alpha1, alpha2, beta1, and gamma1 chains as mediators of sickle cell adhesion. The monoclonal antibody 4C7, which binds at or near the G-domain of the laminin alpha5 chain, significantly inhibited sickle RBC adhesion. These results suggest that an adhesive region for sickle RBCs is contained within the laminin alpha5 chain.     

The kinetics of bioaccumulation of zinc, copper, lead and cadmium by oysters (Crassostrea iredalei and C. belcheri) under tropical field conditions

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC-FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2-84.9% at concentrations of 10 micrograms/l. The R.S.D. values were 1.7-3.9% (n = 8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.     

Gas chromatograph injection liner for continuous analyte admission into a mass spectrometer

An inexpensive modification to a gas chromatography injector liner is reported that facilitates continuous admission of analyte into a gas chromatograph/mass spectrometer (GC/MS) for methods development. The MS methods development liner can be made by making simple modifications to commercially available liners and fits into standard injectors in place of the normal liners without any need to break vacuum in the MS. The injector temperature and gas flow rates are adjusted to provide appropriate analyte levels in the MS, which can be admitted under conditions identical with those of real analyses, including co-admission of column bleed. The device is particularly useful for development of tandem MS methods in GC/MS/MS instruments, which are configured with the GC as the sole sample inlet.